Low-dose dopaminergic drugs to delay the progression of spinocerebellar ataxia type 3 and alzheimer's disease

Abstract

The present invention refers to dopaminergic drugs such as dopamine precursors and aromatic L-amino acid decarboxylase (AADC) inhibitors for use in a treatment to delay the progression of pathologies in which abnormal amyloid deposits spread to different brain regions, consisting of Spinocerebellar Ataxia Type 3 and Alzheimer's Disease, administered in levodopa-equivalent doses below 300 mg / day. The present invention further refers to pharmaceutical formulations comprising the said dopaminergic drugs. The present invention's compounds pharmaceutical formulations and uses may be advantageously employed in a treatment to prevent further accumulation of amyloid deposits in neurons, and minimize adverse effects associated with the prolonged use of dopamine precursors and / or their peripheral degradation.

Description

[0001] DESCRIPTION LOW-DOSE DOPAMINERGIC DRUGS TO DELAY THE PROGRESSION OF SPINOCEREBELLAR ATAXIA TYPE 3 AND ALZHEIMER’S DISEASE Technical field of the invention The present invention relates to the technical field of humanhealth; medical science; preparations for medical purposes; inparticular, medicinal preparations containing organic active ingredients. State of the art Spinocerebellar Ataxia Type 3 (SCA3) and Alzheimer’s Disease (AD) are two neurodegenerative disorders characterized by the deposition in different brain regions of amyloid aggregates containing disease-specific proteins. SCA3, or Machado-JosephDisease, is a rare disease that was first recognized in the 1970sin families of Azorean descent. SCA3 symptoms start to manifest on average between the third to fifth decade of life and include progressive lack of coordination / accuracy of movement (cerebellar ataxia), abnormal gait and impaired balance, involuntary muscle contractions (dystonia), stiffness of muscles (spasticity), difficulties of speech (dysarthria), swallowing disorders, and double vision. Healthy individuals harbour CAG repeat lengths between 12 and 44, whereas in SCA3-affected individuals this length is consensually above 61. The expansion of the unstable CAG repeat is translated into an expanded polyglutamine (polyQ) tract of ataxin-3, a protein whose accumulation in neuronal nuclearinclusions is a pathological hallmark of SCA3. SCA3 belongs to thegroup of polyQ diseases that includes five additional SCA types and Huntington’s disease (HD), among others. SCA3 also belongs to the large group of protein-misfolding diseases that include Alzheimer’s disease (AD) and are characterized by the aggregation and accumulation of misfolded proteins in cells. AD, in particular, is characterized by the accumulation of two types of protein aggregates consisting of (i) extracellular senile plaques containing the peptide amyloid-beta (Abeta) and (ii) intracellular neurofibrillary tangles composed of the protein tau. The accumulation of Abeta plaques affects the cerebral cortex and hippocampus areas responsible for cognition language processing, reasoning and memory consolidation, whereas tau tangles typically spread across the medial temporal lobe, followed by the association cortices and eventually reaching the isocortical regions [1]. Familial and sporadic forms of AD have been described. Approximately 1–2% of the total AD cases are inherited in an autosomal dominant fashion, showing alterations in specific genes such as the amyloid precursor protein gene [2]. AD is the most common neurodegenerative disease and represents 60 to 80% of all cases of dementia. While the onset of amyloid deposition can be ascribed to a combination of multiple factors, it is well known that the occurrence of amyloid deposits of ataxin-3 in SCA3, or Abeta and tau in AD, can accelerate disease progression due to the formationof new (and smaller) aggregates at the surface of pre-existingones. Among the different therapeutic strategies to tackle protein misfolding diseases, abnormal protein aggregation can be targeted by delaying or preventing (i) the de novo formation of new assemblies, (ii) the growth or elongation of existing aggregates and (iii) the templated formation of new assemblies. The simultaneous inhibition of the three steps is also possible through upstream strategies that prevent the protein misfolding events preceding protein self-assembly. No disease-modifying treatments are available for SCA3. Phase I clinical trials have addressed the safety of using antisense oligonucleotides to lower ataxin-3 levels in SCA3 patients (NCT05160558). This approach relies on the hypothesis that ataxin- 3 is not an essential protein for cell function; conversely, protein aggregation inhibition strategies aim at preventing toxic gain-of-function without affecting the functional roles of the (non-aggregated) protein. Biohaven reported in May 2022 no effect on the primary outcome of a Phase 3 trial of troriluzole for SCA3. Repurposing trehalose for the treatment of SCA3 is under investigation by Seelos Therapeutics (NCT05490563). On the other hand, anti-amyloid strategies have been approved for the treatment of AD using monoclonal antibodies directed against Abeta. Examples of these immunotherapies include Aducanumab (Aduhelm, Biogen) and Lecanemab-irmb (Leqembi, Eisai / Biogen). These drugs lower the amount of Abeta in the brain but it is not consensual whether this effect improves people’s symptoms and quality of life. Potential side effects associated with Aducanumab and Lecanemab-irmb require regular brain imaging tests to monitor e.g. brain swelling and bleeding spots. In a statement dated January 2024, Biogen discontinued Aducanumab ‘to reprioritize its resources in AD’ and not for reasons related to safety or efficacy. In Parkinson’s disease (PD), the death of dopaminergic neurons leads to reduced levels of dopamine and the consequential loss of controlled body movements, tremors, slowness of movements (bradykinesia), muscle rigidity and balance problems. While there is no cure for PD, dopamine replacement therapies, such as the use of dopaminergic drugs, help to manage PD symptoms. Dopamine is a catecholamine neurotransmitter mainly produced in the substantia nigra and ventral tegmental area of the midbrain, but also in the hypothalamus, amygdala, and cortex regions of the brain. Severe dopamine deficiency in the striatum is responsible for the manifestation of the first motor symptoms in PD [3]. To combat these symptoms, dosage regimes of 100 mg levodopa 3 times daily (300 mg / day) are typically prescribed at the time the disease is diagnosed [4]. Levodopa drug formulations are available in combination with AADC inhibitors (such as carbidopa or benserazide) and COMT inhibitors (such as entacapone and tolcapone). Alternatively, levodopa-sparing strategies are adopted in the medical management of PD, including the dopamine agonists apomorphine HCl, rotigotine, pramipexole and ropinirole, or the selective monoamine oxidase (MAO) inhibitors selegiline and rasagiline [5]. Independently of their mechanism of action, antiparkinsonian drugs can be prescribed in “dopamine-equivalentdoses” as a way to standardize treatment regimens [6].Dopamine-like agents are known to have an anti-amyloid activity, manifested, for example, by the destabilization / disaggregation of Abeta fibrils in the presence of high concentrations of dopamine (above 100 mM)

[0019] ; the exact mechanism behind this anti-amyloid activity is, however, unclear. Dopamine also prevents the formation of amyloid fibrils of α-synuclein (asyn), a protein whose misfolding and aggregation are associated with PD. In this case, the inhibitory effect of dopamine results from the formation ofdopamin-asyn adduct and the consequential aggregation into non-fibrillar asyn oligomers [7,8]. Nevertheless, to face theprogressive dopamine deficiency in PD, dopaminergic drugs are administered at gradually increasing doses to PD patients. For example, the dopamine precursor levodopa is typically dosed 3 times daily but, as PD progresses, higher and more frequent doses are required to combat the slowed response to medication [4]. Levodopa is frequently combined with aromatic L-amino acid decarboxylase (AADC) inhibitors, such as carbidopa, which prevent peripheral dopamine synthesis, thereby increasing the amount of levodopa that reaches the brain. However, dopamine overproduction by the peripheral metabolism is on the basis of adverse effects of levodopa, such as nausea and vomiting. High doses of levodopa are also associated with severe dyskinesia symptoms (fast,unpredictable, and involuntary movements) and behaviouralcomplications [9]. As such, despite anti-aggregation effects of dopamine anddopamine-like agents are documented at molecular level, knowledgeabout the effective clinical utility of dopaminergic drugs in the setting of protein aggregation diseases other than PD is totallylacking in prior art. Furthermore, clinical evidence supportingthe administration dose and formulation parameters to reacheffectiveness while minimizing adverse effects in conditions otherthan PD is totally lacking. Summary of the InventionThe present invention stems from the original finding thatdopamine, at sub-stoichiometric dopamine / polypeptide ratios,effectively inhibits the formation of further protein assembliesof ataxin-3 and Abeta. Thus, for these specific targets, theintended inhibitory effect can be achieved by correcting a milddopamine deficiency. As such, herein disclosed is administrationof <300 mg / day levodopa for use in a treatment to inhibit the propagative steps of ataxin-3 and Abeta aggregation and, in this way, slow down the progression of Spinocerebellar Ataxia Type 3 (SCA3) and Alzheimer’s Disease (AD), respectively. Clinical evidence from individuals enrolled by the Clinical Research Consortium for Spinocerebellar Ataxias the NationalAlzheimer Coordinating Center (NACC) supports that even inpatients in which Parkinsonian symptoms are not manifested,levodopa-based therapies are associated with beneficial effects onSCA3 and AD, which is not observed for dopamine-replacementtherapies such as the use of dopamine agonists. Low-dose (<300mg / day) levodopa formulations are herein disclosed that containAADC inhibitors present in higher amounts relative to levodopa than what is currently known in the art, with the advantage of lesser secondary effects. As such, the present invention refers to levodopa for use in thetreatment of neurodegenerative amyloid deposit diseases consistingof SCA3 and AD, through stimulation of mild dopamine production inthe brain and inhibition of pathogenic protein aggregates, whereinlevodopa is administered in a dose below 300 mg / day, according toclaim 1. The present invention further refers to a pharmaceutical formulation for use in the treatment of neurodegenerative amyloid deposit diseases consisting of SCA3 and AD, through stimulation of mild dopamine production in the brain and inhibition of pathogenicprotein aggregates, comprising levodopa and other agents selectedfrom the list consisting of AADC inhibitors, COMT inhibitors, prodrugs, biodegradable polymers that facilitate levodopa delivery and combinations thereof, according to claim 2. In another embodiment, in the pharmaceutical formulation mentionedabove, the said AADC inhibitors comprise carbidopa or benserazide,in the AADC inhibitor:levodopa proportion (in mass) of 1:n, where n is any number below 4, according to claim 3. In another embodiment, in the pharmaceutical formulation mentioned, the said COMT inhibitors comprise entacapone ortolcapone, in the COMT inhibitor:levodopa proportion (in mass) of1:k, where k is any number between 0.1 and 1, according to claim 4. Detailed description of the Invention A seeded-aggregation, Thioflavin-T (ThT) fluorescence assay is developed in which the aggregation of 10 µM Abeta is elicited by the presence of 0.5 µM of pre-formed fibrils of Abeta. Using the new biophysical assay, >1200 drug repurposing compounds previously tested during ataxin-3 aggregation

[0010] were now screened for highly potent inhibitors of seeded Abeta aggregation. Surprisingly, dopamine hydrochloride, carbidopa, methyldopa, methyldopate hydrochloride, apomorphine hydrochloride hemihydrate, dobutamine hydrochloride, luteolin and racepinephrine HCl are confirmed as inhibitors of nonseeded ataxin-3 aggregation and seeded Abeta aggregation (Figures 1A and 1B). By studying the seeded aggregation of Abeta, potent inhibitors could be identified using compound:Abeta ratios as low as 0.2:1 (Figure 1B). Taking together, the high potency of these inhibitors during the aggregation of both ataxin-3 and Abeta suggest common mechanisms of action in which the templated formation of new assemblies is likely targeted. The used concentrations of inhibitor (2 µM) is, for the particular case of dopamine hydrochloride, within the normal dopamine levels reported for brain regions such as the hippocampus (up to 10 µM) or the hypothalamus (up to 10 µM)

[0011] . The dose-response study performed for dopamine hydrochloride during Abeta aggregation points to a half-maximal inhibitory concentration of IC50=0.51 µM (Figure 2B), which is within the normal dopamine levels reported for the substantia nigra (up to 1 µM) and prefrontal cortex (up to 1 µM)

[0011] . To further elucidate the effect of low-concentrationdopamine hydrochloride on the final product of Abeta aggregation, morphology (Figure 3), size (Figure 4), and physicochemical properties (Figure 5) of Abeta assemblies is analyzed at the end of the seeded aggregation assay. Using Transmission Electron Microscopy (TEM), large bundles of Abeta fibrils are identified in the absence of dopamine (Figure 4, left) but not in the presence 5 µM dopamine (Figure 4, right). By Dynamic Light Scattering (DLS) analysis, a population with large sizes (hydrodynamic radius RH > 100 nm) and another with small sizes (RH < 100 nm) is found. In both cases, characterized by lower RH values when 10 µM dopamine is present (Figure 4). Lastly, using the filter retardation assay, the final products of Abeta aggregation in the presence of dopamine are found to be less SDS-resistant than the control sample (Figure 5). Among the identified compounds, dopamine hydrochloride is one of the most potent inhibitors of ataxin-3 and Abeta aggregation. Although dopamine does not readily cross the blood-brain barrier (BBB), dopamine brain levels can be safely increased through the administration dopaminergic drugs such as the dopamine precursor levodopa. Based on the newly identified role of low-concentration dopamine in modulating the seeded aggregation of Abeta, it can be reasoned that increasing the levels of dopamine may prevent the spreading of large Abeta assemblies but cannot prevent AD onset nor completely revert AD progression.Overall, the above mentioned findings disclosed that mild dopaminedysfunction causes accelerated and more extensive proteinaggregation in the brain of SCA3 and AD patients. To restore healthy levels of brain dopamine, SCA3 and AD patients with mild dopamine deficiency need lower doses of dopaminergic drugs than those used to treat PD patients with severe dopaminedeficiency. Such treatment delays SCA3 or AD progression, whilethe secondary effects possibly associated with the use of antiparkinsonians in SCA3 or AD are minimized.Clinical evidence comes from analysis of two independent cohorts:(i) individuals with SCA3 and also SCA1, 2, 6, 7, 8, and 10enrolled by the Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA) and (ii) (ii) cerebrospinal fluid (CSF) biomarker information anddisease progression rates included in the National Alzheimer Coordinating Center (NACC) Uniform Data Set for subjects with normal cognition (NC), mild cognitive impairment (MCI) and dementia (DE).Examination of these data to investigate how the progressions ofSCA3 and AD are affected by the exposition to dopaminergic drugsby individuals with concomitant Parkinsonian symptoms demonstratesdopamine-restoring therapies, such as the use ofcarbidopa / levodopa (CA / LA), are associated with beneficial effectsthat are not observed for dopamine-replacement therapies, such asthe use of dopamine agonists. Despite adverse effects are also identified in connection to the long-term use of CA / LA, they are not found in the long-term use of other antiparkinsonians. In the case of the CRC-SCA cohort, analysis focuses on individuals with all spinocerebellar ataxias (SCAs) whose ataxia progressionis assessed by the Scale for the Assessment and Rating of Ataxia(SARA) between 2010 and 2023; to account for important covariates affecting the time evolution of SARA scores, SCA3 was limited tocases with genetic and age information available (Figure 6). Among the group of 716 individuals with SCAs with monitored SARA scores, 58 (8.1%) have taken CA / LA formulations, whereas 26 (3.6%) have taken other antiparkinsonian drugs, mainly dopamine receptor agonists. To visualize the overall effects of CA / LA and other antiparkinsonian medications, the time required for major diseaseprogression events to occur in cases and controls was computed.These events are defined as corresponding to an increase in the SARA score larger than 7 points relative to the initial assessment. Kaplan-Meier plots of event probability vs. time suggest that the CA / LA group has a lower susceptibility to a major disease decline over the 6 years that follow the initial ataxia assessment. This apparent advantage is lost in the post-6-year period (Figure 7A). Conversely, the group of other antiparkinsonians shows no major differences to controls over the initial 6 years and appears toslightly improve in the subsequent period (Figure 7B). To accountfor the confounding factors affecting disease progression, analysis is centered on the most represented disease (SCA3) and on participants with known age and CAG repeat length. A linear mixedmodel is employed to evaluate the effect of CA / LA expositionadjusted for covariates, thus taking full advantage of a SARA score dataset in which participants with a single medical visit are alsoincluded. Through multivariate analysis, significant effects ofage, time since the first visit, CAG repeat length and CA / LA treatment are identified from the measured data of SARA score progress at 6 and 13 years (Figure 8 and Table 1). As expected, age, time and repeat length are characterized by positive values of the LME coefficient β (Table 1), meaning that SARA scores increase with the covariate magnitude. For CA / LA, the interaction term confirms a statistically significant effect in favour of drug exposition (negative β) both at 6 and 13 years. Such an effect is not observed for other antiparkinsonians (Figure 9 and Table 2). In the case of the NACC, a multivariate analysis of AD biomarker data (Figure 10) and a survival analysis of disease progressiondata (Figure 11) are performed. The multivariate analysis uses CSFlevels of A^42, total tau (T-tau), and tau phosphorylated at threonine 181 (P-tau) that are measured using enzyme-linked immunosorbent assay (ELISA) or Luminex multiplex xMAP assay protocols. The disease progression analyses use cognitive decline data obtained after clinical evaluation or the information of deathevents reported to NACC. CA / LA use is associated with lower levelsof A^42, T-tau and P-tau in the CSF of subjects diagnosed at the baseline with NC, MCI or DE (Figure 12, top). Although the populations of cases and controls are heterogeneously affected by different covariates, the lowering effect of CA / LA on CSF biomarker levels is confirmed by LME model regressions adjusted for the effects of variables ‘age’, ‘sex’ and ‘presence of the APOE ε4 allele’ (Table 3, top). Statistically significant effects are also invariably observed for the APOE covariate in decreasing CSF A^42 levels and increasing CSF T-tau and P-tau. The effects of variable ‘age’ in increasing CSF T-tau and P-tau are statistically significant in all subgroups except DE. In our study, the variable ‘sex’ had no significant effect on the biomarker levels except for CSF A^42 in the DE subgroup (CSF A^42 levels are lower in females diagnosed with DE). The percentages of subjects with reported Parkinsonian symptoms in the CA / LA cases / control groups are27.4% / 0.5% (NC), 32.2% / 5.0% (MCI) and 14.7% / 6.7% (DE). In subjectsprescribed dopamine agonists or other dopaminergic drugs distinct from CA / LA, the correspondence between lowered levels of ADbiomarkers and CA / LA use is analysed. None of the effects reportedfor CA / LA are observed in the groups subject to levodopa-sparingtreatments. No significant effect of non-CA / LA antiparkinsonians is identified on the levels of CSF T-tau and P-tau in any of the subgroups NC, MCI or DE, while the CSF A^42 levels are increased (rather than decreased) in one of the subgroups (Figure 12,bottom). A multivariate analysis adjusted for variables ‘age’,‘sex’ and ‘presence of the APOE ε4 allele’ is performed to accountfor the heterogeneous populations of cases and controls (Table 3,bottom). Again, APOE Ɛ4 carriers have systematically lower CSFA^42 and higher T-tau and P-tau levels, while older subjects of the subgroups NC and MCI tend to have higher T-tau and P-tau levels; no evident effect is identified for variable ‘sex’. The percentages of subjects with reported Parkinsonian symptoms in thenon-CA / LA cases / control groups is 3.7% / 4.0% (NC), 30.8% / 3.6% (MCI)and 12.2% / 6.0% (DE). Despite the observation of lower CSF A^42levels suggests changes in the A^ metabolism, the T-tau- and P-tau-lowering effects might implicate reduced neurodegenerationand, consequently, slower cognitive decline. To confirm this, allcases with reported use of CA / LA are assessed – including thosewithout CSF biomarker data available – and a population of controls(without reported use of CA / LA) is defined upon automatic 1:1matching by ‘age’, ‘sex’ and ‘APOE ε4 allele presence’. The Kaplan-Meier method is then used to determine the cumulative probabilitiesof cognitive decline from NC to MCI / DE (Figure 13A) or from MCI to DE (Figure 13B). According to the logrank Mantel-Cox tests thatare performed, CA / LA exposition does not change the cognitivedecline in NC subjects but increases the probability of no cognitive decline in MCI subjects (P=0.03; logrank hazard ratio1.321 [95% confidence interval 1.020 to 1.710]). The beneficialeffects of CA / LA can also affect life expectancy. Kaplan-Meiercurves are generated using the dates of deaths reported during theobservation period (2005-2024) for the same NC and MCI populationsthat are assessed for cognitive decline. An analogous procedure isadopted for the subjects with baseline DE who were prescribed CA / LA (cases) and respective controls matched by ‘age’, ‘sex’ and ‘APOE ε4 allele presence’. In all these analyses, the probability ofsurvival to death events is never increased in CA / LA-exposed groups(Figure 14). Survival rates decrease in the NC subgroup (Figure 14A, P=0.002; logrank hazard ratio 0.50 [95% confidence interval 0.334 to 0.763]) and remain unaltered in the MCI (Figure 14B) andDE (Figure 14C) subgroups. The decreased survival rates inindividuals with NC exposed to CA / LA might be solely because thecontrol group is less affected by PD. To rule-out this possibility,the survival probabilities for individuals with NC, MCI and DE exposed to non-CA / LA antiparkinsonians and matched controls wascomputed(Figure 15). No significant differences are identified, although the computed survival curves point to increased survival rates for non-CA / LA cases after ~5 years of monitoring (Figure 15A). This analysis demonstrates that the currently availableCA / LA antiparkinsonians – but not other antiparkinsonians – areassociated with lower survival rates by individuals with NC. It has been reported that adverse effects associated with long- term use of levodopa start to manifest in PD 6-7 years after the onset of Parkinson’s symptoms

[0012] . Therefore, CA / LA treatments may benefit SCA3 and AD patients already showing aggravating signs of the disease, in line with the apparent delays (i) in SARA score increase achieved for SCA3 patients (Figure 8) and (ii) in cognitive decline achieved for MCI subjects (Figure 13B), or (iii) the lowered levels of CSF biomarkers observed in the NACC data set(Figure 12, top). Such a therapeutic strategy balances (i) theincreased risk of major disease decline identified in CA / LA- treated individuals with SCAs (Figure 7A), and (ii) the increased risk of death that was identified in CA / LA-treated NC patients (Figure 14A). In summary, even if Parkinsonian symptoms are not manifested by most SCA3 or AD patients, levodopa-based drugs with an adjusted dose to treat mild dopamine deficiency are clinically useful to combat SCA3 and AD progression at the expense of lesser secondaryeffects. The daily doses of AADC inhibitors required to preventthe peripheral synthesis of dopamine from levodopa are, in the case of carbidopa, >75 mg, and, in the case of benserazide, >100 mg [13,14]. These limits are imposed by the amount of extracerebral decarboxylase that needs to be inhibited in adults. The required doses of AADC inhibitors are warranted by levodopa-based formulations containing CA:LA ratios or benserazide:LA ratios of 1:4; as the daily levodopa dose is increased, CA:LA ratios of 1:10 are alternatively adopted to avoid excessive carbidopa intake

[0013] . In the present invention, however, daily doses of levodopabelow those prescribed during early PD are disclosed for use inthe treatment of SCA3 and AD. In one embodiment of the present invention, low-dose levodopa pharmaceutical formulation comprises >75 mg / day carbidopa or >100 mg / day benserazide. To comply with this condition, in other embodiments, low-dose levodopa-based formulations are characterized by ratios of decarboxylase inhibitor:LA of, for example, 1:3 or 1:2.In another embodiment the pharmaceutical composition comprises aprodrug and / or biodegradable polymers that facilitate levodopa delivery on a more continuous basis.In certain embodiments the said pharmaceutical composition isadministered by the oral route. In other embodiments the saidpharmaceutical composition is administered by non-oral routes, including intestinal gels, inhaled formulations, and subcutaneous routes.Example: Pharmaceutical composition comprising a dopamineprecursor such as levodopa at daily doses below 300 mg / day In accordance with one embodiment, a subject is administered a pharmaceutical composition comprising a dopamine precursor such as levodopa at daily doses below 300 mg / day. In one embodiment the administered pharmaceutical additionally comprises compounds selected from the group consisting of an AADC inhibitor (AADCi) such as carbidopa and / or a COMT inhibitor (COMTi) such as entacapone in the proportions levodopa:AADCi:COMTi of 1:n:k, where n is any number below 4, and k is any number between 0.1 and 1. Brief description of the Figures Figure 1. Screening of drug-repurposing compounds during the aggregation of (A) 3 µM ataxin-3 and (B) 10 µM Abeta in the presence of pre-formed fibrils of Abeta (0.5 µM). Symbols: ThT fluorescence increase measured for each tested compound (2 µM final concentration) at the end of (A) 30 h or (B) 48 h incubation. Different colors indicate the four 384-well microplates used to screen 1280 drug-repurposing compounds. Common hits to the ataxin- 3 and Abeta screenings are identified by the circled numbers nextto each open square: 1 – apomorphine hydrochloride hemihydrate; 2– dobutamine hydrochloride; 3 – carbidopa; 4 – methyldopa; 5 –luteolin; 6 – dopamine hydrochloride; 7 – racepinephrine HCl; 8 –methyldopate hydrochloride. These hits are either catecholaminessuch as dopamine or have a catechol structure similar to that ofdopamine. For the screening of ataxin-3 aggregation inhibitors, the protocol previously developed by us

[0014] was adopted with minor modifications. Briefly, ataxin-3 was expressed using the pDEST17ATX3(13Q) plasmid transformed into E. coli BL21(DE3)-SI cells (Life Technologies) and then purified as previously described

[0013] . Ataxin-3 aggregation was monitored by monitoring thioflavin T (ThT) fluorescence increase at 480 nm (440 nm excitation) on a CHAMELEON V plate reader (HIDEX) using a 384-well microplate (low flange, black, flat bottom, polystyrene; Corning, Kennebunk, ME). 50 μL samples of 3 μM ataxin-3 in aggregation buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 1 mM DTT) containing 15 μM ThT were incubated at 37 ºC, and ThT fluorescence was measured over time. To prevent evaporation, 30 μL of paraffin oil was put in each reaction well with an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120). A commercial chemical library (Prestwick, Illkirch) containing 1280 drug repurposingcompounds was screened. Ataxin-3 was dispensed into a 384-wellmicroplate with an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120, Eppendorf, Germany). Then an automated liquid handler (JANUS Automated Workstation; PerkinElmer) equipped with pin tool replicators (V&P Scientific) coupled to a Modular Dispense Technology (MDT) head was used to add 0.1 μL of test compounds (from a 1 mM stock) or DMSO (for controls). A total of four 384-well microplates were filled, each plate testing 320 chemical compounds (1 compound per well) and running 32 control reactions in the presence of DMSO. The final reaction mixture contained 3 μM of ataxin-3 and 2 μM of test compound / DMSO. The selection of hit compounds was based on the increase of fluorescence signal at the end of 30 h incubation. For Abeta modulators screening, we adapted the protocol used during the unseeded aggregation of ataxin-3 to consider the seeded aggregation of Abeta. Briefly, depsi-Aβ (1–42) click peptide (Genscript, ref. RP10017) was dissolved in 0.1% TFA to a final concentration of 200 μM (stock solution) and stored at ^80 °C until use. The peptide concentration was confirmed by UV absorption measurements at 280 nm using a Tyr extinction coefficient of 1490 M^1cm^1. Before experiments, the depsi-Aβ (1–42) peptide was thawed and converted into Aβ(1-42) (Abeta) by changing from acidic pH (0.1% TFA, pH 2.5) to pH > 7.4. For this, the stock solution of depsi Aβ (1–42) peptide was diluted 1:1 using 200 mM phosphate buffer pH 7.5. Abeta seeds were produced by incubating 900 μL samples of 50 μM Abeta on a Thermo shaker incubator (MSC-100 Cooling Thermoshaker Incubator, Labgene Scientific) at 37 ^C and 1000 rpm. After 8 days of incubation, the samples were sonicated for 2 min (SONOPULS HD2200, Bandelin Electronic, probe: MS73) during on / off sequences of 15 seconds each (10% max power). The Abeta seeds thus produced were frozen in liquid nitrogen and kept at ^80 °C. To study the seeded aggregation of Abeta, 50 μL samples of 10 μM Abeta in aggregation buffer (100 mM phosphate buffer pH 7.5, 0.2 mM EDTA and 0.02% NaN3) containing 20 μM ThT are incubated at 37 ºC with 0.5 µM Abeta seeds, and ThT fluorescence is measured over time. To prevent evaporation, 30 μL of paraffin oil is put in each reaction well with an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120). Compound screening was performed as described for ataxin-3 using a commercial library of 1280 drug repurposing compounds and automated liquid handler to add the aggregation assay components and pin tool replicators to add 0.1 μL of test compounds or DMSO. The final reaction mixture contained 10 μM of Abeta, 0.5 µM Abeta seeds and 2 μM of test compound / DMSO. The selection of hit compounds was based on the increase of fluorescence signal at the end of 48 h incubation. Figure 2. Dose-response analyses of the effect of dopamine on theaggregation of (A) µM ataxin-3 and (B)10 µM Abeta in the presenceof 0.5 µM pre-formed Abeta fibrils. (A) Symbols: influence of dopamine concentration on the lag phase duration measured during the aggregation of ataxin-3 with a homorepeat segment containing 13 glutamines (13Q, blue) or 77 glutamines (77Q, red). Solid lines: numerical fit by a concentration-response curve (IC50 values of 0.11 µM and 0.62 µM for the 13Q and 77Q variants, respectively). Dashed lines: Visual reference. (B) Symbols: influence of dopamineconcentration on the ThT increase measured at the end of 48 h. Theobtained IC50 values suggest secondary inhibition mechanisms for which only substoichiometric concentrations of the inhibitor are required to delay the replication of previously existing aggregates. For the studies of dopamine dose response during ataxin-3 aggregation, we used the same ataxin-3 concentration as before (3 µM). A stock solution of dopamine is prepared at 40 µM and a series of serial-diluted concentrations are made to obtain 12 different tested concentrations: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02 and 0.01 µM dopamine. Experiments for each condition are run in triplicate. For control experiments, the same scheme is adopted departing from a 40 µM DMSO stock solution. All serial dilutions are prepared using an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120, Eppendorf, Germany). To determine the half-maximal inhibitory concentration (IC50), a four parameter Hill equation was fitted to 30 h-ThT-fluorescence increase vs. dopamine concentration data. For the studies of dopamine dose response during Abeta aggregation, we used the same Abeta and seeds concentrations as before (10 µM and 0.5 µM, respectively). A stock solution of dopamine is prepared at 40 µM and a series of serial-diluted concentrations made to obtain 10 different tested concentrations: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 and 0.04 µM dopamine. Experiments for each condition are run in triplicate. For control experiments, the same scheme is adopted departing from a 40 µM DMSO stock solution. To determine the IC50, a four parameter Hill equation was fitted to 48 h-ThT-fluorescence increase vs. dopamine concentration data. Figure 3. TEM images after negative staining of Abeta aggregates produced after 72 h incubation of 10 µM Abeta and 0.5 µM Abeta seeds in the absence (left) and presence (right) of 5 µM dopamine hydrochloride. Samples were adsorbed onto glow-discharged, carbon- coated films supported on 300-mesh nickel grids and negatively stained with 1% (w / v) uranyl acetate. Grids were visualized using a JEM-1400 (JEOL) TEM at an accelerating voltage of 80 kV. For the TEM, DLS and filter retardation analyses, the aggregation buffer used during the seeded aggregation assay of Abeta was slightly changed in relation to the one used for compound screening and dose response studies. To avoid uranyl phosphate mineralization in TEM analysis, HEPES was used instead of phosphate in the aggregation buffer (final composition: 100mM HEPS pH 7.5, 0.2 mM EDTA and 0.02% NaN3). In addition, in these assays, the stock solutions of dopamine hydrochloride were prepared in water. Figure 4. DLS analysis of the effect of dopamine hydrochloride on the end-product of Abeta aggregation. Size distributions by intensity measured after 120 h incubation of 10 µM Abeta and 0.5 µM Abeta seeds in the absence and presence of 5 µM dopamine hydrochloride. Samples (300 µl) were incubated in a UV-Cuvettemicro cuvette (Brand, Germany) with a cap, maintained at 37 ^Cwithout shaking and analysed in a Zetasizer Nano ZS DLS system (Malvern Instruments). Figure 5. Filter retardation assay of the endpoint samples of Abeta aggregation produced after 120 h incubation of 10 µM Abeta and 0.5 µM Abeta seeds in the absence and presence of 5 µM dopamine hydrochloride. Samples incubated for 5 minutes in TBS without (-) and with (+) 2% SDS (95 ^C) were filtered through a cellulose acetate membrane (0.2 µm, Whatman, Buckinghamshire, UK) pre- equilibrated in TBS, and the membrane was washed twice with TBS supplemented with 0.1% (w / v) SDS. The membrane was removed from the filter apparatus and blocked with 5% (w / v) non-fat dry milk in TBST overnight at 4 ^C. SDS-resistant Abeta fibrils retained in the membrane were probed with monoclonal anti-Abeta antibody BAM- 10 (Sigma, A3981) 1:2,000 for 2 h at room temperature (RT). Anti-mouse IRDye^ 800CW (LI-COR) 1:20,000 was used as secondaryantibody. Detection was performed by Infrared fluorescence using an Odyssey® CLx Infrared Imaging system. Figure 6. Identification of SCA cases and controls. *Early symptomatic: cases that, simultaneously, have a baseline SARA score lower than 8 and a difference between the last SARA score and the baseline lower than 3. This study is based on the CRC-SCA cohort of subjects with SCA1, 2, 3, 6, 7, 8 and 10 which were voluntarily recruited from 12 centers in the US [3]. The CRC-SCA dataset we assessed contains anonymized information collected from 2010 to 2023 on ataxia severity, CAG repeat expansion, concomitant medication, demographics and comorbidities. The patients seen in the ataxia clinics were referred by the patients themselves, community physicians, local support groups as well as the National Ataxia Foundation. The uniform study protocol was approved by local institutional review boards, and written informed consent was obtained from each participant. The inclusion criteria included (1) diagnosis of the spinocerebellar ataxias in the study subjector his / her affected family members, (2) presence of ataxia, (3)willingness to participate in the study, and (4) the age of 6 years and older. The exclusion criteria were (1) known recessive, X- linked or mitochondrial ataxias, 2) concomitant disorder(s) thataffect the Scale for the Assessment and Rating of Ataxia (SARA)and other ataxia measures, or 3) exclusion of the spinocerebellar ataxias by DNA testing. The study comprises two non-parametric analyses of the population with SCA1, 2, 3, 6, 7, 8 or 10, and one multivariate analysis of the population with SCA3. We selected 716 participants in the CRC-SCA cohort who had documented medication history and SARA scores and who were not considered early- symptomatic. Patients with monitored SARA scores who showed baseline values lower than 8 and a difference between the last SARA score and the baseline lower than 3 were considered early- symptomatic. For the non-parametric Kaplan-Meier analyses, 293 participants who had a single SARA score evaluation were excluded. For the multivariate analysis, only participants with SCA3 were selected (570 excluded out of 716). In the first non-parametric analysis, controls and cases were defined from the medication history of each participant, namely according to the absence (controls) or existence (cases) of reported use of CA / LA medication. This criterion comprises current or past prescriptions of clinical drugs whose names include the words “levodopa”, “carbidopa”, “sinemet”, “sinomet”, “stalevo”, “dopar” or “larodopa”. For the second non-parametric analysis, the population of 382 participants without recorded CA / LA use was further dividedinto those who did (cases) or did not (controls) take any of thefollowing anti-parkinsonians: ”pramipexole”, “ropinirole”, “requip”, “neupro”, “bromocriptine”, “pergolide”, “cabergoline”, “tolcapone”, “rotigotine”, “entacapone”, “rasagiline”, “mirapex”, or “artane”. From the 146 participants included in the multivariate analysis, 122 controls and 24 cases were selected according to the absence (controls) or existence (cases) of reported use of the CA / LA medication defined above. In addition, a larger group of 155 participants (131 controls and 24 cases) that includes early- symptomatic SCA3 gene carriers was analyzed. Figure 7. Resistance to major disease decline in spinocerebellar ataxias: the effect of antiparkinsonians. (A and B) Kaplan-Meier analysis for events of a large SARA score increase (> 7 points) in patients (A) who have previously taken CA / LA compared with controls, and (B) who have previously taken other antiparkinsonians compared with controls. The time from the baseline to the observation of a major disease decline was assessed by the Kaplan-Meier method, where the event of interest corresponds to a SARA score increase larger than 7 points. Kaplan-Meier curves were plotted to compare the probability of event survival for SCA patients with (cases) and without (controls) reported use of CA / LA and other anti- parkinsonians. Figure 8. Effect of exposition to CA / LA on SCA3 evolution. Progress of SARA scores (A) observed in individuals with SCA3 and (B) predicted by the LME model for (top) controls and (bottom) cases of exposition to CA / LA. (B) 95% confidence intervals for 6-year predictions computed assuming a random SCA3 patient, 58 years of age, and expanded allele length 70. To account for confounding factors such as the type of spinocerebellar ataxia, age, and the length of expanded allele repeat, we focused on the most common SCA (SCA3) and selected cases and controls for which genetic and age information was available. A linear mixed-effects (LME) model regression was performed to determine whether there was a significant change in the time evolution of SARA scores upon exposition to CA / LA medication. The adopted Wilkinson notation for the SARA score response was the following: SARA~TREATMENT+TIME+TREATMENT:TIME+AGE+ALLELE+(1|PATIENT), where TREATMENT denotes whether the patient was exposed to CA / LA or not, TIME represents the number of years since the baseline visit, TREATMENT:TIME is the interaction term, AGE and ALLELE are additional covariates computed in years and number length of expanded allele, respectively, and 1|PATIENT denotes random intercepts assigned to individual patients. The model was regressed using the fitlme function of MATLAB R2023a (MathWorks,Natick, MA) to determine LME coefficients (β) and the associated P values. The LME model fitted to the longitudinal SARA increase at 6 years was used to predict the evolution of SARA scores for a 56-year-old patient with an expanded allele length of 56. P values less than 0.05 were considered significant. Figure 9. Effect of exposition to other antiparkinsonians on SCA3 evolution. Progress of SARA scores (A) observed in individuals with SCA3 and (B) predicted by the LME model for (top) controls and (bottom) cases of exposition to non-CA / LA antiparkinsonians. (B) 95% confidence intervals for the 6-year predictions computed assuming a random SCA3 patient with 58 years of age, and expanded allele length 70. To check whether delayed SCA3 evolution is due to the use of other antiparkinsonians, the LME analysis was additionally performed for subjects exposed to non-CA / LA antiparkinsonians and controls. Figure 10. Flow chart of subject selection for multivariate analyses of CSF biomarker data in NC, MCI and DE subgroups exposed to CA / LA or other antiparkinsonians, and controls. The participants in this study are those included in the subset of the NACC-UDS sample (March 2024 data freeze) who allowed the sharing of research data with commercial entities. The NACC program was developed to facilitate collaborative involvement among Alzheimer’s Disease Research Centers (ADCs) in the US. In 2005, ADCs began collecting longitudinal demographic, clinical, neuropsychological, and diagnostic data using version 1 of the data set, which was subsequently updated and expanded with the implementation of versions 2 (2008) and 3 (2015) of the UDS [4,31]. The UDS Version 3 is nonproprietary (available upon a data request) and provides a standardized methodology for assessing cognition and clinical characteristics of patients with AD and other neurological diseases [4]. Each ADC enrols its participants according to its protocols, e.g., through clinician referral, self-referral by participants or family members, and active recruitment in community organizations. This longitudinal protocol requires annual follow-up while the participant is able and willing to be involved, and comprises 8 data-collection forms that are completed by clinicians or clinical staff in each ADC (https: / / naccdata.org). This study is divided into a multivariate analysis of CSF biomarker data and a survival analysis of disease progression data. Cases for which the baseline characterization of prescribed medication occurred more than 2 years after the CSF test were excluded. Results from at least one CSF test are available for 1,942 subjects comprising the clinical subgroups diagnosed at the baseline with NC (972), MCI (293) and DE (677). For each clinical subgroup, CA / LA cases and controls were defined according to the absence (controls) or existence (cases) of reported use of levodopa or carbidopa medication. This criterion comprises current or past prescriptions of clinical drugs whose names include the words ‘levodopa’ or ‘carbidopa’ in any of the 40 ‘DRUG’ fields of UDS form A4. In an additional multivariate analysis, the population of CA / LA controls, i.e., participants who do not have reported use of CA / LA, were further divided into thosewho did (cases) or did not (controls) take any of the followingnon-CA / LA antiparkinsonians: 'pramipexole', 'ropinirole', 'bromocriptine', 'pergolide', 'cabergoline', 'tolcapone', 'rotigotine', 'entacapone', or 'rasagiline'. Each subgroup wascharacterized in terms of number of cases of Parkinsonian symptomsreported as PD, other parkinsonian disorder, Parkinsonian signs, or Parkinsonian gait disorder. This analysis uses CSF levels of A^42, total tau (T-tau), and tau phosphorylated at threonine 181 (P-tau) that are measured using enzyme-linked immunosorbent assay (ELISA) or Luminex multiplex xMAP assay protocols. Figure 11. Flow chart of subject selection for the analyses of cognitve decline by NC and MCI subgroups exposed to CA / LA and controls. In the survival analysis of cognitive decline, subjects diagnosed with NC (13,442) or MCI (6,906) at the baseline were divided into CA / LA cases and controls following the same criterion as in the multivariate analysis above. Then, nearest-neighbour 1:1 matching was performed to obtain equally-sized CA / LA-exposed and CA / LA- naive samples in each NC and MCI groups. Disease progression was also characterized in tems of the probability of death events in the NC and MCI groups, and in an additional group of subjects diagnosed with DE at the baseline. The disease progression analyses use cognitive decline data obtained after clinical evaluation or the information of death events reported to NACC. The main covariates considered in our study are, for each clinical subgroup, age, sex and the presence of APOE allele ^4. Here, ‘APOE ^4 carriers’ classify the presence of APOE ^3 / ^4, ^4 / ^4 or ^4 / ^2 genotypes. We also collected information about the education level (in years), race (grouped as White, Black or African American, and others), comorbidities such as hypertension, cardiovascular disease, and cancer, and concurrent medication such as nonsteroidal anti-inflammatory drugs (NSAIDs), anticoagulants, antipsychotics, hormones, antihypertensives, diabetes medication, lipid-lowering medication, and antidepressants. Figure 12. Association between the use of (top) CA / LA and (bottom) non-CA / LA antiparkinsonians and the levels of CSF biomarkers for AD. Boxplots of measured levels of CSF A^42 (red), P-tau (blue) and T-tau (green) for the (A and D) NC, (B and E) MCI and DE (C and F) subgroups. The boxes extend from the 25th to 75th percentiles, the central line is the median and the whiskers represent minimum and maximum values. Stars show the statistical significance of differences between controls and cases quantifiedby LME model regression, with more stars indicating lower P valuesassociated with the variable ‘TREATMENT’ in Table 3. Number of CSF tests: (A) 1481 controls and 79 CA / LA cases; (B) 377 controls and 54 CA / LA cases; (C) 886 controls and 53 CA / LA cases; (D) 1358 controls and 117 CA / LA cases; (E) 354 controls and 23 CA / LA cases; (E) 812 controls and 73 CA / LA cases. In this analysis, a linear mixed-effects (LME) model regression was performed to determine if the levels of CSF A^42, T-tau and P-tau are significantly affected by CA / LA use in the NC, MCI and DE subgroups. The adopted Wilkinson notation for the CSF levels of each biomarker was the following: BIOMARKER~TREATMENT+AGE+SEX+APOE+(1|PATIENT), where TREATMENT denotes whether the patient was exposed to CA / LA or not, AGE, SEX, and APOE are additional covariates, and 1|PATIENT denotes random intercepts assigned to individual patients. The model was regressed using the fitlme function of MATLAB R2023a (MathWorks, Natick, MA) to determine LME coefficients (β) and the associated P values. Figure 13. Resistance to cognitive decline by participants in the CA / LA-exposed and CA / LA-naive samples. Kaplan-Meier analysis for events of cognitive decline by subjects with (A) NC and (B) MCI. In this analysis, the Kaplan-Meier method was used to determine the cumulative probabilities of cognitive decline from NC to MCI / DE or from MCI to DE. Next, we performed propensity score matching and logrank (Mantel-Cox) tests to quantify the magnitude of the CA / LA effect. For that, the NearestNeighbors.fit() function from the Python package scikit-learn was first used for automatic 1:1 nearest-neighbour matching, and then GraphPad Prism software (La Jolla, CA, USA) was used to determine P values for each comparison of survival curves. Figure 14. Overall survival of participants in the CA / LA-exposed and CA / LA-naive samples. Kaplan-Meier analysis for death events by subjects with (A) NC, (B) MCI and (C) DE. In this analysis, the Kaplan-Meier method was used to determine the cumulative probabilities of the occurrence of death events in each clinical subgroup considering the cases of CA / LA-exposition and controls. Figure 15. Overall survival of participants exposed to non-CA / LA antiparkinsonians and controls. Kaplan-Meier analysis for death events by subjects with (A) NC, (B) MCI and (C) DE. In this analysis, the Kaplan-Meier method was used to determine the cumulative probabilities of the occurrence of death events in each clinical subgroup considering the cases of exposition to non- CA / LA antiparkinsonians and controls. Table 1. Covariate-adjusted effect of CA / LA exposition on SCA3 progression. LME coefficients, 95% confidence intervals and P values fitted to the 13-year and 6-year SARA monitoring data. Table 2. Covariate-adjusted effect of exposition to non CA / LA antiparkinsonians on SCA3 progression. LME coefficients, 95% confidence intervals and P values fitted to the 13-year and 6-year SARA monitoring data. The two-sided unpaired t-test or the Fisher test were carried out using the functions ttest2 or fishertest of Matlab R2023a (Mathworks, Natick, MA) for comparisons between two groups of continuous variables or categorical variables, respectively. Table 3. Covariate-adjusted effect of (top) CA / LA and (bottom) non-CA / LA treatments on the AD biomarkers. LME coefficients, 95% confidence intervals and P values fitted to measured levels of A^42, T-tau and P-tau in the CSF of participants with NC, MCI and DE. Table 4. Characteristics of participants exposed to Antiparkinsonians (CA / LA, non-CA / LA and controls) in the CSF biomarker study. The two-sided unpaired t-test or the Fisher test were carried out using the functions ttest2 or fishertest of Matlab R2023a (Mathworks, Natick, MA) for comparisons between two groups of continuous variables or categorical variables, respectively. Table 5. Characteristics of participants exposed to CA / LA and controls in the analyses of cognitive decline and survival rates. Table 1. Covariate-adjusted effect of CA / LA exposition on SCA3 progression. LME coefficients, 95% confidence intervals and P values fitted to the 13-year and 6-year SARA monitoring data. 13 YEARS EIATATT T 6 YEARS T REATMENT

[0002] Table 2. Covariate-adjusted effect of exposition to non CA / LA antiparkinsonians on SCA3 progression. LME coefficients, 95% confidence intervals and P values fitted to the 13-year and 6-year SARA monitoring data. 13 YEARS EFFECT Lower U erP IATATT T 6

[0003] Table 3. Covariate-adjusted effect of (top) CA / LA and (bottom) non-CA / LA treatments on the AD biomarkers. LME coefficients, 95% confidence intervals and P values fitted to measured levels of A^42, T-tau and P-tau in the CSF of participants with NC, MCI and DE. NC MCIDE Biomarker EFFECT ue1018)8 / 01(0136l / 41ir 401241e2)1 / 01- 10(s 7i 2s 8ir 2i1t 6r 7t 86 4

[0004] Table 4. Characteristics of participants exposed to Antiparkinsonians (CA / LA, non-CA / LA and controls) in the CSF biomarker study. NC VariableMCI DEC ntr l C P v l C ntr l C P v l C ntr l C P v lue32) A4L / A4C(a7podov3el / a7podibra C 7676e138) A L8 / A C9-no1n(sn ia5nos7inkrapit 4n A4reh3t 8453 ep essa . . . . . . . . . 9

[0005] Table 5. Characteristics of participants exposed to CA / LA and controls in the analyses of cognitive decline and survival rates. VariableNC MCI DEControls Cases P value Controls Cases P value Controls Cases P value82271514718869725 References [1] Cai Y, Du J, Li A, Zhu Y, Xu L, Sun K, Ma S, Guo T, for the Alzheimer’s Disease Neuroimaging Initiative. Initial levels β- amyloid and tau deposition have distinct effects on longitudinal tau accumulation in Alzheimer’s disease. Alz Res Therapy 2023;15:30. [2]Long JM, Holtzman DM. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 2019;179:312–339. [3] Chinta SJ, Andersen JK. Dopaminergic neurons. The International Journal of Biochemistry & Cell Biology 2005;37:942– 946. [4] Armstrong MJ, Okun MS. Diagnosis and Treatment of Parkinson Disease: A Review. JAMA 2020;323:548. [5] Goldenberg MM. Medical management of Parkinson’s disease. Pharmacy and Therapeutics 2008;33:590. [6] Jost ST, Kaldenbach M, Antonini A, Martinez-Martin P, Timmermann L, Odin P, Katzenschlager R, Borgohain R, Fasano A, Stocchi F, Hattori N, Kukkle PL, Rodríguez-Violante M, Falup- Pecurariu C, Schade S, Petry-Schmelzer JN, Metta V, Weintraub D, Deuschl G, Espay AJ, Tan E, Bhidayasiri R, Fung VSC, Cardoso F, Trenkwalder C, Jenner P, Ray Chaudhuri K, Dafsari HS, the International Parkinson and Movement Disorders Society Non-Motor Parkinson Disease Study Group. Levodopa Dose Equivalency in Parkinson’s Disease: Updated Systematic Review and Proposals. Movement Disorders 2023;38:1236–1252. [7] Conway KA, Rochet J-C, Bieganski RM, Lansbury PT. Kinetic Stabilization of the α-Synuclein Protofibril by a Dopamine-α- Synuclein Adduct. Science 2001;294:1346. [8] Lee H-J, Baek SM, Ho D-H, Suk J-E, Cho E-D, Lee S-J. Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers. Exp Mol Med 2011;43:216. [9] De Bie RMA, Clarke CE, Espay AJ, Fox SH, Lang AE. Initiation of pharmacological therapy in Parkinson’s disease: when, why, and how. The Lancet Neurology 2020;19:452–461.

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Claims

CLAIMS1. Levodopa for use in the treatment of neurodegenerativeamyloid deposit diseases consisting of Spinocerebellar Ataxia Type 3 (SCA3) and Alzheimer’s Disease, with or without Parkinsonian symptoms, through stimulation of mild dopamine production in the brain and inhibition of pathogenic protein aggregates, wherein levodopa is administered in a dose below 300 mg / day.

2. Pharmaceutical formulation for use in the treatment ofneurodegenerative amyloid deposit diseases consisting of Spinocerebellar Ataxia Type 3 (SCA3) and Alzheimer’s Disease, through stimulation of mild dopamine production in the brain and inhibition of pathogenic protein aggregates, comprising levodopa and other agents selected from the list consisting of AADC inhibitors, COMT inhibitors, prodrugs, biodegradable polymers that facilitate levodopa delivery and combinations thereof.

3. Pharmaceutical formulation according to claim 2, whereinsaid AADC inhibitors comprise carbidopa or benserazide, in the AADC inhibitor:levodopa proportion (in mass) of 1:n, where n is any number below 4.

4. Pharmaceutical formulation according to claim 2, whereinsaid COMT inhibitors comprise entacapone or tolcapone, in theCOMT inhibitor:levodopa proportion (in mass) of 1:k, where kis any number between 0.1 and 1.Lisbon, 5th December 20241

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